Receptor-mediated endocytosis (RME) is the process of membrane invagination by which cell surface receptors are selectively internalized into acidic intracellular compartments. This is the major cellular route for uptake of macromolecular drugs, such as antibody-drug conjugates and liposome encapsulated drugs. Delivery of drugs into cells by this pathway confers greater specificity of action of the drug by virtue of its sequestration inside the cell. Furthermore, the metabolism of the drug can be affected by exposure to the acidic conditions in subcellular compartments. thus RME is an important cellular process that influences both the pharmacodynamics and pharmacokinetics of macromolecular drugs and its molecular control should be understood. RME occurs as a result of polymerization of clathrin molecules on the cytoplasmic face of the plasma membrane. The polymerized clathrin forms a polyhedral network of protein that traps cell surface receptors and causes their internalization along with bound ligands. the size of the clathrin network appears to influence the size of the particles internalized and may explain the limits observed for liposome uptake in different cell types. the aims of this proposal are to define the structural features of the clathrin molecule that control and polymerization reaction. One experimental approach involves expressing fragments of the clathrin heavy chain subunit and the complete light chain subunits in bacteria. Expression and mutation of the individual light chain subunits will allow mapping functional regions, such as the calcium binding site that influences clathrin assembly. Co-expression of clathrin light chains and heavy chain fragments will be used to study the interaction of the clathrin subunits. This approach could also lead to the expression and purification of large amounts of light chains assembled with heavy chain fragments to attempt crystallographic studies. The main obstacle to crystallographic analysis of the clathrin molecule has been the heterogeneity of the light chain subunits. In another approach to structural analysis, clathrin will be purified from cells that have recently been produced in the laboratory that express only one form of clathrin light chain. This homogeneous population of clathrin will also be analyzed for assembly properties. Finally, cells will be transfected with clathrin fragments and mutated subunits that will be expressed in levels to compete with endogenous proteins and thereby study the effect of these molecules on clathrin assembly and liposome uptake in situ. Understanding the molecular basis for clathrin polymerization will provide insight into processes that control the uptake of macromolecular drugs.